Deoxynucleotide triphosphates (dNTPs) are the natural raw materials for, or substrates of, viral and cellular polymerases, and reverse transcriptases. In the absence of dNTP, cells and viruses cannot synthesize their genetic materials and, consequently, cell division and/or virus replication is blocked. Sufficient quantities of dNTP must be available at the time of DNA synthesis if DNA replication or reverse transcription is to take place.
Ribonucleotide reductase (RR) is the sole enzyme responsible for the synthesis of dNTP. Without RR, dNTP production is slowed or completely eliminated, because the salvage of dNTP from cellular DNA represents only a minor, insignificant source of dNTP. It has been recognized for some time that hydroxyurea (HU) inhibits cellular, bacterial, and viral RR. HU as well as other RR-inhibitors have been used as antiviral and anticancer drugs (Cory, J. G. [1988] Adv. Enzyme Regul. 27:437-455). HU was also recently shown to block the replication of HIV by the same mechanism which limits the substrate (dNTP) for viral reverse transcriptase (Lori, et al. [1994] Science 266:801-805).
Cellular RR is a highly regulated enzyme. It consists of two dissimilar subunits M1 and M21 which are independently regulated during cell division. In cells resting in G0-G1 (no cellular DNA synthesis), cellular RR activity is diminished. Therefore, neither DNA nor RNA viruses can efficiently synthesize DNA in such quiescent cells. Some viruses, such as herpes simplex virus (HSV), pseudorabies, and Varicella-Zoster Virus (VZV), however, carry RR in their genome. These viral RR catalyze the same reaction as the mammalian enzyme, but the viral enzyme is free from allosteric regulation. Consequently, large pools of dNTP are created for viral DNA synthesis. Therefore, these viruses are able to productively replicate not only in dividing cells but also in quiescent cells even through such cells do not produce dNTP.
Typically, retroviruses can only infect dividing cells because dividing cells have sufficient amounts of dNTP available to support reverse transcription, while non-dividing cells have insufficient amounts of this substrate. The only known exceptions are the lentiretoviruses (e.g., HIV-1 and SIV). Some virus variants infect terminally differentiated cells, e.g. macrophages (MF). MF have higher levels of dNTP which are sufficient from macrophage tropic HIV or SIV variants to reverse transcribe their RNA genome (Gao et al. [1993] PNAS 90:8923-8928). However, these viruses do not replicate in quiescent lymphocytes because of incomplete reverse transcription of the viral RNA to DNA.
Retrovirus vectors are candidates for foreign gene delivery and have been evaluated for use in gene therapy approaches. Retrovirus vectors can permanently modify cells by integrating a foreign gene into the genome of a target cell. Retrovirus vectors are also advantageous because they are generally safe; therefore, they have already been approved for experimental gene therapy.
There are two major problems hindering the successful use of retrovirus vectors in gene therapy: the low efficiency of gene transfer and the incompetence to transduce non-dividing (quiescent) cells. Recent improvements of retrovirus vectros include the design of vectors with an extended host range (based, for example, on vesicular somatitis viruses instead of murine leukemia viruses) (Bums et al. PNAS 90:8033-8037), vectors which use multiple receptors for entry (Miller and Chen [1996] J. Virol. 70:5564-5571), vectors that are resistant to human serum (Cosset et al. [1995] J. Virol 69:7430-7436), and vectors which target new surface proteins (Somia et al. [1995] PNAS 92:7570-7574). Chu T. H. et al. [1994] Gene Therapy 1 (5):292-299) constructed a chimeric virus envelope of retrovirus vector with an antigen binding site of an antibody. Nilson et al. (Nilson, B. H. et al. [1996] Gene Therapy 3(4):280-286) exploited protease-substrate interaction for viral targeting. They fused epidermal growth factor (EGF) to a retroviral envelope and showed binding and gene transfer into cells expressing an EGF-receptor.
Lieber et al. ([1995] Hum. Gene Ther. 6:5-11) reported that adenovirus-mediated transfer of the amphotropic retrovirus receptor cDNA increased retroviral transduction in cultured cells. Adenovirus-mediated gene transfer results in permanent modification of cells not only with the viral-receptor gene but also with several adenovirus-vector genes. Introduction of undesired new genes into cells for the purpose of increasing the transfer of a therapeutic gene is highly undesirable and may be dangerous in gene therapy. Curiel et al. (Cold Spring Harbor Gene Therapy Meeting 1996, p. 158) reported using adenovirus vectors for gene delivery. They accomplished cell specific gene delivery by modifying the tropism of adenoviruses by introduction of ligands into the viral-vector envelope. Similarly, the coat protein of adenovirus vector particles were modified by other investigators to increase the entry in different cell types (Curiel et al., supra, p. 159).
All of the above approaches address transfer of genes into dividing cells. None of the presently known retrovirus vectors transduce quiescent cells. Naldini et al. [1996] Science 272:263-267) considered the use of a lentiretrovirus, HIV-1, as a gene delivery vehicle into non-dividing, quiescent cells. The HIV-based vector was able to deliver the lac-Z gene into growth-arrested cells. However, this approach did not result in integration and gene expression in growth-arrested cells.